Many hormones and neurotransmitters exert their actions on target cells by raising the levels of cAMP, which activates cAMP-dependent protein kinase. Activation of this kinase results in the phosphorylation of molecules which produces a variety of effects including the alteration of catalytic properties of enzymes, changes in the conductance of ion channels, and modification in the levels of expression of various genes. The effects produced upon a given hormone or neurotransmitter acting through cAMP depend on the cell type and the developmental stage of the cell. This specificity is due to the selective expression of only a few of all the possible substrates for cAMP-dependent protein kinase in any given cell. In order to define the mechanisms and specific functions of cAMP-mediated signal transduction in a particular cell type, it is necessary to identify the substrates for cAMP-dependent protein kinase in these cells.
An example of an extensively studied phosphoprotein is the dopamine and cAMP-regulated phosphoprotein DARPP-32 which is expressed in the cells of the rat brain caudate putamen that also express dopamine D.sub.1 receptors (Walaas, S. I. (1983) Nature 301:69-71). The phosphatase-1 inhibitory function of DARPP-32 is dependent on its state of phosphorylation, which is regulated by receptor stimulation. Dopaminergic (D.sub.1) and glutamatergic (NMDA) receptor stimulation both regulate the extent of DARPP-32 phosphorylation but in opposite directions. Dopamine D.sub.1 receptor stimulation enhances cAMP formation resulting in the phosphorylation of DARPP-32, and phosphorylated DARPP-32 is a protein phosphatase-1 inhibitor. Glutamatergic NMDA receptor stimulation elevates intracellular calcium, leading to activation of calcineurin and dephosphorylation of phospho-DARPP-32, and reducing the phosphatase-1 inhibitory activity of DARPP-32 (Hemmings, H. C. (1984) Nature 310: 503-505, Halpain, S. (1990) Nature 343: 369-372). Therefore the normal levels of phosphorylated DARPP-32 in brain cells are dependent on a balance between the excitatory and inhibitory receptors and their respective neurotransmitters.
Another cAMP-regulated phosphoprotein which is highly enriched in the caudate putamen is ARPP-16. A related phosphoprotein, ARPP-19, was identified because it copurified with ARPP-16, has identical phosphorylation sites, and cross reacts with antibodies raised to ARPP-16 (Horiuchi, A. (1990) J Biol Chem 265 (16):9476-84). The amino acid sequences of ARPP-16 and ARPP-19 are identical except for an additional 16 amino acids at the NH.sub.2 -terminal of ARPP-19. The expression of these highly similar phosphoproteins has been examined in the rat and human brain and in peripheral organs (Girault, J. A. (1990) J Neurosci 10 (4):1124-1133, and Brene, S. (1994) J Neurosci 14 (3):985-998). ARPP-16 and ARPP-19 are present in the caudate putamen, globus pallidus, cerebral cortex, substantia nirga and nucleus accumbens, but ARPP-16 levels were found to be 2-3 times higher than ARPP-19 levels in the caudate putamen. ARPP-16 was only observed in the brain regions listed above and not in any peripheral organs, while ARPP-19 was found in all brain regions and in all peripheral organs tested.
Stimulation of the cAMP pathways has been found to enhance survival, differentiation, and mitosis in cultured neuroblasts, indicating that the cAMP-dependent pathways have a role in the regulation of neuronal development (Pincus, D. W. (1990) Brain Res 514: 355-357). Similarly, tissues that are not terminally differentiated such as a variety of tumor-derived cell lines contained ARPP-19 in similar or greater levels than normal tissue, while ARPP-16 was not found in any of them (Girault, supra).
The developmental expression of ARPP-16 and ARPP-19 has been studied in mouse brain and peripheral organs. ARPP-16 appears at the end of the first post-natal week in brain structures, increases until eight weeks in the caudate putamen, and then plateaus, while ARPP-19 levels are highest in embryo tissues and decrease with development in all regions examined (Girault, supra). In spite of the high degree of sequence identity between these two molecules these results indicate that they have different and very distinct functions; ARPP-19 is associated with cells that are not fully differentiated and plays a role in more ubiquitous cell development processes while ARPP-16 is specifically expressed in brain tissues that express dopamine receptors.
Many disorders of movement, such as Parkinson's and Huntington's diseases, have been attributed to disturbances of the basal ganglia structures within the brain including the caudate putamen. These diseases involve selective loss of specific neurons which results in an imbalance between excitatory and inhibitory neurotransmittors and their receptors. Current drug therapies attempt to restore this balance, but until the mechanisms of these disease processes are more fully understood, more effective treatments will not be available. The discovery of the polynucleotide sequence encoding the novel human phosphoprotein protein associated with cell development, regulation of neurotransmitters, and signal transduction presents the opportunity to investigate the mechanisms of diseases associated with these processes. Discovery of molecules related to a novel human phosphoprotein protein would satisfy a need in the art by providing a new means for the diagnosis, prevention, treatment, or study of degenerative brain diseases and abnormal cell death and proliferation.